118 Chapter 5 Live Tracing of Non-Viral Vector-Based Fluorescence Expression in hiPSC-CMs Based on our data, it appears that the cell cycle-related effects of CHIR99021 are the most crucial for the transfection efficiency in hiPSC-CMs (Figure 4). To demonstrate that indeed the active cycling hiPSC-CMs were to express the mCherry plasmid first, we recorded timelapse videos in bright-field and immunofluorescence mode for 12 h starting at 1-day posttransfection of the cells. After 24 h and 36 h of transfection we observed a pattern of mainly neighboring cells that started to express the mCherry fluorescent protein (Figure 5A). Quantification of neighboring cells confirmed that 75.25% were paired mCherry positive cells, while 14.35% of mCherry positive cells were unpaired and 10.4% were binucleated cells (Figure 5B). Next, we analyzed our time-lapse videos for cells that showed a rolled-up morphology during mitosis and found that the fraction of cells undergoing cytokinesis started to express mCherry protein mainly right after cytokinesis had occurred (Figure 5C). In cells that became binucleate after mitosis we noticed an expression of mCherry in both nuclei upon completion of the binucleation process (Figure 5D). These data indicate that incorporation of vectors occurs before or during M-phase, while expression of the PF256-H2B-mCherry vector starts immediately after the M-phase. \ Figure 5. Time-lapse analysis of non-viral vector mCherry incorporation in proliferating hiPSC-CMs. (A) Representative time-lapse images of the mCherry positive hiPSC-CMs 12 h and 24 h post-transfection with mCherry-H2B plasmid and ViaFect transfection reagent. (B) Quantification of the mCherry positive paired, unpaired and binucleated cells represented in percentages (n = 100). Data are shown as mean ± SEM. *** p < 0.001 as calculated by Student’s t-test. (C) Selected time-lapse images visualizing mCherry expression in a mononuclear hiPSC-CM undergoing cytokinesis. (D) Selected time-lapse images demonstrating mCherry expression after binucleation of a mononuclear hiPSC-CM. Time is indicated in hours (h). Scale bar indicates 50 µm.
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